The cytotoxic effect of aloin A and B was performed with Vero-E6 using PrestoBlue™ Cell Viability kit. The assay was carried out as per the manufacturer’s protocol. Briefly, around 20,000 cells were seeded for 6h in 96 well plates. Then the cells were refreshed with 100µl of media (EMEM with 10%FBS and 1% of Pen/Strep) along with 50 and 100µM aloin A and B and incubated for 24 and 48h at 37°C respectively. PrestoBlue solution (10µl) was added and continued the incubation for 1h at 37°C. The absorbance was taken at 530/25 excitation and 590/35nm emission using Bio-Tek Synergy HT fluorescent plate reader.
Synergy ht fluorescent plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy HT fluorescent plate reader is a versatile instrument designed for a range of fluorescence-based assays. It is capable of detecting fluorescent signals in multi-well plate formats, enabling researchers to perform high-throughput analysis of samples.
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5 protocols using synergy ht fluorescent plate reader
1
Cytotoxicity Evaluation of Aloin A and B
2
Quantifying Adherent Cell Viability
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PC3 and DU145 cells (5 × 104 cells/100 μL) were seeded into each well of a 96 well plate and were incubated for 30 minutes at 37°C after which the wells were washed with PBS three times and incubated with 1 μmol/L Calcein AM (Invitrogen Life Technologies, USA) for 3 minutes. The cells that attached to the plate were quantified by measuring the fluorescence intensity at 458/528 nm in each well on a Synergy HT fluorescent plate reader (BioTeK, USA). All experiments were performed in triplicate.
3
Nitric Oxide Quantification Assay
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This assay was performed following protocols described in (Lewis et al., 2016 ) with slight modifications. Cultures were grown in modified DSM 205 medium, and at 24 h growth, a 5 ml aliquot was harvested by centrifugation at 3901 g for 10 min. Cells were resuspended in 1 ml Halo‐HBSS with 10 µM DAF‐FM diacetate and incubated statically at 37°C for 60 min. Cells were then pelleted (12,000 g for 5 min) and washed once in fresh Halo‐HBSS. Cells were finally resuspended in 700–900 µl of Halo‐HBSS to adjust for cell density differences, triplicate aliquots (200 µl) of stained cell suspensions were transferred to black optically clear‐bottom 96‐well plates (Costar 3904), and both OD600 and fluorescence (relative fluorescent units, RFU) were measured as described in (Lewis et al., 2016 ) using a Biotek Synergy HT fluorescent plate reader.
4
Ovarian Cancer Cell Cytotoxicity Assay
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Trypsinized ovarian cancer cells (A2780, SKOV3, and A2008) were seeded at approximately 20,000 per well into a 96-well plate. The cells were allowed to recover from trypsinization for 24 hours. They were then treated with various dilutions of the GluN1 antagonist MK-801 (100–800 µM, n=8) and an IgG preparation of PANN1 antibodies precipitated from serum with 50% saturated (NH4)2SO4 (1:5,000, 1:2,500, 1:1,000, 1:500, 1:100, 1:50, 1:25, and 1:10 relative to the original serum concentration, n=8) for 72 hours. A2780 cells were treated with concentrations from 100 to 800 µM of the GluN2B antagonist ifenprodil over 48 hours (n=16). The Alamar Blue cell viability reagent was added to each well at a concentration of 10% and incubated at 37°C for 1–4 hours or until the substrate had changed to a pink color. The plate was read on a Synergy HT fluorescent plate reader (BioTek Instruments Inc., Winooski, VT, USA) at a fluorescence excitation wavelength of 570 nm and emission wavelength of 590 nm. The cell treatments were performed in quadruplicate, and each experiment was performed five times. Cell viability was normalized to untreated cell values and expressed as a percentage of those values.
5
Cytotoxic Effects of Phytochemicals on Vero-E6 Cells
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The cytotoxic effect of selected phytochemicals were carried out with Vero-E6 cells using PrestoBlue™ Cell Viability kit as described earlier (Lewis et al., 2022 (link)). Briefly, Vero-E6 cells (20,000 cells) were seeded overnight in 96 well plates. Then, 100 µL of complete media (EMEM+10% FBS+1%Pen/Strep) was added to refresh the cells along with 50, 100, and 200 µM selected phytochemicals, and incubation was continued for 24, 48, and 72 hs at 37°C respectively. Cytotoxicity detection regent (PrestoBlue solution) was added and incubated for additional 1h at 37°C. The absorbance was taken at 530/25 excitation and 590/35 nm emission using Bio-Tek Synergy HT fluorescent plate reader.
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